Recombinant DNA vaccine encoding multiple domains related to inhibition of neurite outgrowth: a potential strategy for axonal regeneration

Myelin-derived proteins, such as tenascin-R (TN-R), myelin associate glycoprotein (MAG), and Nogo-A, inhibit the CNS regeneration. By targeting specifically the inhibitory epitopes, we have investigated whether vaccination with a recombinant DNA molecule encoding multiple domains of myelin inhibitors may be useful in CNS repair. We show here that the recombinant DNA vaccine is able to activate the immune system but does not induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Importantly, it promotes axonal regeneration in a spinal cord injury model. Thus, the application of DNA vaccine, encoding multiple specific domains of major inhibitory proteins and/or their receptors, provides another promising approach to overcome the inhibitory barriers during CNS regeneration.     

Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury

Axon regeneration after injury to the adult mammalian CNS is limited in part by three inhibitory proteins in CNS myelin: Nogo-A, MAG, and OMgp. All three of these proteins bind to a Nogo-66 receptor (NgR) to inhibit axonal outgrowth in vitro. To explore the necessity of NgR for responses to myelin inhibitors and for restriction of axonal growth in the adult CNS, we generated ngr(-/-) mice. Mice lacking NgR are viable but display hypoactivity and motor impairment. DRG neurons lacking NgR do not bind Nogo-66, and their growth cones are not collapsed by Nogo-66. Recovery of motor function after dorsal hemisection or complete transection of the spinal cord is improved in the ngr(-/-) mice. While corticospinal fibers do not regenerate in mice lacking NgR, regeneration of some raphespinal and rubrospinal fibers does occur. Thus, NgR is partially responsible for limiting the regeneration of certain fiber systems in the adult CNS.     

A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with a retained intron 4 induces change in mitochondrial morphology and growth arrest

Upon spinal cord injury, the myelin inhibitors, including the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp), bind to and signal via a single neuronal receptor/co-receptor complex comprising of Nogo receptor 1(NgR1)/LINGO-1 and p75 or TROY, impeding regeneration of injured axons. We employed a cell-free system to study the binding of NgR1 to its co-receptors and the myelin inhibitor Nogo-A, and show that gangliosides mediate the interaction of NgR1 with LINGO-1. Solid phase binding assays demonstrate that the sialic acid moieties of gangliosides and the stalk of NgR1 are the principal determinants of these molecular interactions. Moreover, the tripartite complex comprising of NgR1, LINGO-1 and ganglioside exhibits stronger binding to Nogo-A (Nogo-54) in the presence of p75, suggesting the gangliosides modulate the myelin inhibitor-receptor signaling.     

Characterization of myelin ligand complexes with neuronal Nogo-66 receptor family members

Nogo, MAG, and OMgp are myelin-associated proteins that bind to a neuronal Nogo-66 receptor (NgR/NgR1) to limit axonal regeneration after central nervous system injury. Within Nogo-A, two separate domains are known interact with NgR1. NgR1 is the founding member of the three-member NgR family, whereas Nogo-A (RTN4A) belongs to a four-member reticulon family. Here, we systematically mapped the interactions between these superfamilies, demonstrating novel nanomolar interactions of RTN2 and RTN3 with NgR1. Because RTN3 is expressed in spinal cord white matter, it may have a role in myelin inhibition of axonal growth. Further analysis of the Nogo-A and NgR1 interactions revealed a novel third interaction site between the proteins, suggesting a trivalent Nogo-A interaction with NgR1. We also confirmed here that MAG binds to NgR2, but not to NgR3. Unexpectedly, we found that OMgp interacts with MAG with a higher affinity compared with NgR1. To better define how these multiple structurally distinct ligands bind to NgR1, we examined a series of Ala-substituted NgR1 mutants for ligand binding activity. We found that the core of the binding domain is centered in the middle of the concave surface of the NgR1 leucine-rich repeat domain and surrounded by differentially utilized residues. This detailed knowledge of the molecular interactions between NgR1 and its ligands is imperative when assessing options for development of NgR1-based therapeutics for central nervous system injuries.     

中枢神经系统轴突再生抑制蛋白

中枢神经系统 (CNS)轴突再生的主要障碍之一是存在抑制再生的蛋白 ,迄今 ,已在少突胶质细胞 /髓鞘中相继发现至少三个重要的轴突再生抑制蛋白 ,即髓鞘相关糖蛋白 (MAG)、Nogo A和少突胶质细胞 /髓鞘糖蛋白 (OMgp)。最近的研究又证实 ,这三个不同的抑制成分可能主要通过与一个共同的受体Nogo6 6受体 (NgR)结合而发挥作用。这些研究成果扩充了对CNS损伤后轴突再生障碍的理解 ,也为探讨CNS损伤的治疗新策略提供了新的思路。     

Bone marrow stromal cells stimulate neurite outgrowth over neural proteoglycans (CSPG), myelin associated glycoprotein and Nogo-A

In animal models, transplantation of bone marrow stromal cells (MSC) into the spinal cord following injury enhances axonal regeneration and promotes functional recovery. How these improvements come about is currently unclear. We have examined the interaction of MSC with neurons, using an established in vitro model of nerve growth, in the presence of substrate-bound extracellular molecules that are thought to inhibit axonal regeneration, i.e., neural proteoglycans (CSPG), myelin associated glycoprotein (MAG) and Nogo-A. Each of these molecules repelled neurite outgrowth from dorsal root ganglia (DRG) in a concentration-dependent manner. However, these nerve-inhibitory effects were much reduced in MSC/DRG co-cultures. Video microscopy demonstrated that MSC acted as “cellular bridges” and also “towed” neurites over the nerve-inhibitory substrates. Whereas conditioned medium from MSC cultures stimulated DRG neurite outgrowth over type I collagen, it did not promote outgrowth over CSPG, MAG or Nogo-A. These findings suggest that MSC transplantation may promote axonal regeneration both by stimulating nerve growth via secreted factors and also by reducing the nerve-inhibitory effects of the extracellular molecules present.     

中枢神经再生抑制因子及免疫治疗研究

成体哺乳动物中枢神经损伤后早期轴突再生失败的一个主要原因是由于髓磷脂抑制分子的存在。Nogo、髓磷脂相关糖蛋白以及少突胶质细胞髓磷脂糖蛋白等神经再生抑制因子的发现,大大促进了中枢神经再生分子机制的研究。它们均能独立通过Nogo-66受体产生对轴突再生的抑制效应,髓磷脂抑制分子及其信号转导机制的研究日益成为中枢神经再生的研究热点,髓磷脂及其信号转导分子特别是Nogo-66受体、p75神经营养素受体成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制上述抑制因子及相关受体NgR或p75NTR可能有助于中枢神经损伤的修复,围绕这些抑制因子及其相关受体介导的信号转导途径,人们提出了多种治疗中枢神经损伤的新思路,其中免疫学方法尤其受到关注。     

Recombinant DNA Vaccine Against Inhibition of Neurite Outgrowth Promotes Functional Recovery Associated with Endogeous NGF Expression in Spinal Cord Hemisected Adult Rats

Axonal regeneration across the site of spinal cord lesion is often aborted in adult mammalian species. The use of DNA vaccine to nullify the inhibitory molecules has been shown to be effective in promoting axonal regeneration in injured spinal cord. The possible molecular mechanisms, however, remain to be elucidated. The present study showed that the administration of recombinant DNA vaccine encoding multiple domains, Nogo-66, Nogo-N, TnR, and MAG, significantly improved hindlimb locomotor functions in rats subjected to ablation of the dorsal halves of the cord. Western blot analysis demonstrated that nerve growth factor (NGF) levels in the spinal cord of immunized rats were significantly upregulated than those of control rats. Immunohistochemistry as well as in situ hybridization confirmed that NGF was expressed in neurons of the spinal cord. These findings indicated that functional recovery in immunized rats could be correlated with endogeous NGF expression in hemisected rat spinal cords. Y. Zhang and C.-G. Hao contributed equally to this work.     

New Insights on Neuronal Alterations in Jimpy Mutant Brain

In spite of abundant data on oligodendrocyte abnormalities in dysmyelinated jimpy brain, little is known about the axonal damage and the expression of neuronal genes. Recent findings indicate that Nogo-A, oligodendrocyte-myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) inhibit axonal growth by binding a common receptor, the Nogo-A receptor (NgR)-p75 complex. In order to evaluate neuronal modifications in the absence of myelin and in the presence of abnormal oligodendrocytes at different developmental stages, the expression of these inhibitory proteins and their receptors was investigated in jimpy mutant brain. Despite the decrease in oligodendrocyte number at P15 and P25 in jimpy, Nogo-A and OMgp mRNA levels are not significantly different compared with control, suggesting an overexpression of neuronal Nogo-A and OMgp in mutant. Double immunolabeling for Nogo-A and neurofilaments shows strong axonal staining of Nogo-A in jimpy and its down-regulation in oligodendrocytes. The current data raise questions about functions of Nogo-A other than neurite growth inhibition in the CNS. No significant changes in NgR mRNA levels were observed in jimpy, where the increase in p75 level can be correlated with the cell death of oligodendrocytes. In the paranodal region, the cell adhesion molecule neurofascin glial isoform NFN155 mRNA level is reduced by 40% whereas neuronal form NFN186 is up-regulated. These results may explain the failure of paranodal region organization, even with normal level of CASPR (paranodin) mRNA detected in jimpy brain.     

The Expression Patterns of Nogo-A,Myelin Associated Glycoprotein and Oligodendrocyte Myelin Glycoprotein in the Retina After Ocular Hypertension

Nogo-A, a major myelin inhibitory protein, inhibits axon growth and synaptic function in the central nervous system. Glaucoma is a progressive neuropathy as a result of retinal ganglion cell (RGC) death. Synaptic degeneration is thought to be an early pathology of neurodegeneration in glaucoma and precedes RGC loss. Here experimental ocular hypertension model was induced in adult rats with laser coagulation of the episcleral and limbal veins. The expression of Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) in the retina was investigated using immunohistochemistry and Western blotting. We found that Nogo-A was expressed in the RGCs and upregulated after the induction of ocular hypertension. OMgp was only expressed in the inner plexiform layer. There was no MAG expression in the retina. Our data provided, for the first time, the expression patterns of three myelin proteins in the adult retina and suggested an important role of Nogo-A in the RGC death and synaptic degeneration in glaucoma.     

The Nogo receptor, its ligands and axonal regeneration in the spinal cord; A review

At least three proteins present in CNS myelin, Nogo, MAG and OMgp are capable of causing growth cone collapse and inhibiting neurite outgrowth in vitro. Surprisingly, Nogo and OMgp are also strongly expressed by many neurons (including neocortical projection cells). Nogo expression is increased by some cells at the borders of CNS lesion sites and by cells in injured peripheral nerves, but Nogo and CNS myelin are largely absent from spinal cord injury sites, which are none the less strongly inhibitory to axonal regeneration. Nogo is found on growing axons during development, suggesting possible functions for neuronal Nogo in axon guidance. Although Nogo, MAG and OMgp lack sequence homologies, they all bind to the Nogo receptor (NgR), a GPI-linked cell surface molecule which, in turn, binds p75 to activate RhoA. NgR is strongly expressed by cerebral cortical neurons but many other neurons express NgR weakly or not at all. Some neurons, such as DRG cells, respond to Nogo and CNS myelin in vitro although they express little or no NgR in vivo which, with other data, indicates that other receptors are available for NgR ligands. NgR expression is unaffected by injury to the nervous system, and there is no clear correlation between NgR expression by neurons and lack of regenerative ability. In the injured spinal cord, interactions between NgR and its ligands are most likely to be important for limiting regeneration of corticospinal and some other descending tracts; other receptors may be more important for ascending tracts. Antibodies to Nogo, mainly the poorly-characterised IN-1 or its derivatives, have been shown to enhance recovery from partial transections of the spinal cord. They induce considerable plasticity from the axons of corticospinal neurons, including sprouting across the midline and, to a limited extent, regeneration around the lesion. Regeneration of corticospinal axons induced by Nogo antibodies has not yet been demonstrated after complete transections or contusion injuries of the spinal cord. It is not clear whether antibodies against Nogo act on oligodendrocytes/myelin or by binding to neuronal Nogo, or whether they can stimulate regeneration of ascending axons in the spinal cord, most of which express little or no NgR. Despite these uncertainties, however, NgR and its ligands offer important new targets for enhancing plasticity and regeneration in the nervous system.     

Spinal axon regeneration induced by elevation of cyclic AMP

Myelin inhibitors, including MAG, are major impediments to CNS regeneration. However, CNS axons of DRGs regenerate if the peripheral branch of these neurons is lesioned first. We show that 1 day post-peripheral-lesion, DRG-cAMP levels triple and MAG/myelin no longer inhibit growth, an effect that is PKA dependent. By 1 week post-lesion, DRG-cAMP returns to control, but growth on MAG/myelin improves and is now PKA independent. Inhibiting PKA in vivo blocks the post-lesion growth on MAG/myelin at 1 day and attenuates it at 1 week. Alone, injection of db-cAMP into the DRG mimics completely a conditioning lesion as DRGs grow on MAG/myelin, initially, in a PKA-dependent manner that becomes PKA independent. Importantly, DRG injection of db-cAMP results in extensive regeneration of dorsal column axons lesioned 1 week later. These results may be relevant to developing therapies for spinal cord injury.     

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.     

The Nogo-66 receptor family in the intact and diseased CNS

The Nogo-66 receptor family (NgR) consists in three glycophosphatidylinositol (GPI)-anchored receptors (NgR1, NgR2 and NgR3), which are primarily expressed by neurons in the central and peripheral mammalian nervous system. NgR1 was identified as serving as a high affinity binding protein for the three classical myelin-associated inhibitors (MAIs) Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), which limit axon regeneration and sprouting in the injured brain. Recent studies suggest that NgR signaling may also play an essential role in the intact adult CNS in restricting axonal and synaptic plasticity and are involved in neurodegenerative diseases, particularly in Alzheimer's disease pathology through modulation of β-secretase cleavage. Here, we outline the biochemical properties of NgRs and their functional roles in the intact and diseased CNS.     

Netrin-1 signaling for sensory axons

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a ‘waiting period’ of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.     

Identification of a functional epitope of the Nogo receptor by a combinatorial approach using ribosome display

The Nogo receptor (NgR) plays a central role in mediating growth-inhibitory activities of myelin-derived proteins, thereby severely limiting axonal regeneration after injury of the adult mammalian central nervous system (CNS). The inhibitory proteins Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) all bind to the extracellular leucine-rich repeat (LRR) domain of NgR, which provides a large molecular surface for protein-protein interactions. However, epitopes within the LRR domain of NgR for binding Nogo, MAG and OMgp have not yet been revealed. Here, we report an evolutionary approach based on the ribosome display technology for detecting regions involved in ligand binding. By applying this method of "affinity fingerprinting" to the NgR ligand binding domain we were able to detect a distinct region important for binding to Nogo. Several residues defining the structural epitope of NgR involved in interaction with Nogo were subsequently confirmed by alanine scanning mutagenesis.     

Generation of an OMgp allelic series in mice

The very limited ability to regenerate axons after injury in the mature mammalian central nervous system (CNS) has been partly attributed to the growth restrictive nature of CNS myelin. Oligodendrocyte myelin glycoprotein (OMgp) was identified as a major myelin‐derived inhibitor of axon growth. However, its role in axon regeneration in vivo is poorly understood. Here we describe the generation and molecular characterization of an OMgp allelic series. With a single gene targeting event and Cre/FLP mediated recombination, we generated an OMgp null allele with a LacZ reporter, one without a reporter gene, and an OMgp conditional allele. This allelic series will aid in the study of OMgp in adult CNS axon regeneration using mouse models of spinal cord injury. The conditional allele will overcome developmental compensation when employed with an inducible Cre, and allows for the study of temporal and tissue/cell type‐specific roles of OMgp in CNS injury‐induced axonal plasticity. genesis 47:751–756, 2009. © 2009 Wiley‐Liss, Inc.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Rho与中枢神经系统轴突再生

Rho是小分子质量GTP酶Rho家族成员,在细胞的一些信号转导途径中起着分子开关的作用.Rho能通过作用于肌动蛋白骨架系统引起轴突生长锥塌陷,从而抑制轴突生长.研究表明,Nogo-A、MAG、OMgp等髓鞘源性的轴突再生抑制分子均可通过激活Rho介导的信号转导途径抑制轴突再生.